[1,2,4]三嗪并[3,4-h][1,8]萘啶-8-酮-7-羧酸衍生物的设计合成及抗菌和抗细胞增殖活性

引用本文

GAO Liu-zhou, LI Tao, XIE Yu-suo, HUANG Wen-long, ZHAO Hui, HU Guo-qiang. Design, synthesis, antibacterial and anti-cell proliferation activities of [1,2,4]triazino[3,4-h][1,8]naphthyridine-8-one-7-carboxylic acid derivatives[J]. Acta Pharm Sin, 2015, 50(3): 332-336. 复制到剪切板

高留州, 李涛, 谢玉锁, 黄文龙, 赵辉, 胡国强. [1,2,4]三嗪并[3,4-h][1,8]萘啶-8-酮-7-羧酸衍生物的设计合成及抗菌和抗细胞增殖活性[J]. 药学学报, 2015, 50(3): 332-336. 复制到剪切板

[1,2,4]三嗪并[3,4-h][1,8]萘啶-8-酮-7-羧酸衍生物的设计合成及抗菌和抗细胞增殖活性

高留州¹, 李涛¹, 谢玉锁¹, 黄文龙², 赵辉¹, 胡国强¹

1. 河南大学化学生物学研究所, 河南开封 475001;
2. 中国药科大学新药研究中心, 江苏南京 210009

收稿日期: 2014-10-08;修回日期: 2014-10-23.

基金项目：国家自然科学基金资助项目(20872028,21072045).

* 通讯作者：Tel/Fax: 86-371-23880680, E-mail: hgqxy@sina.com.cn

摘要：为发现新结构氟喹诺酮先导物, 基于药效团拼合药物设计原理, 用[1,2,4]三嗪核为稠合环, 设计合成了新三环氟喹诺酮衍生物[1,2,4]三嗪并[3,4-h][1,8]萘啶-8-酮-7-羧酸目标化合物 (5a～5p), 结构经光谱数据和元素分析确证, 并对其体外抗菌和抗细胞增殖活性进行了评价。结果表明, 目标化合物对耐药菌株和SMMC-7721肝癌细胞株有较强的抑制活性。构-效关系显示, 供电子基取代的苯基化合物有利于提高抗菌活性, 而吸电子基取代的苯基化合物显示出较强的抗肿瘤活性, 其中部分化合物的IC₅₀值与对照药阿霉素相当。因此, 三嗪稠合的三环氟喹诺酮羧酸作为新的抗菌抗肿瘤新先导物值得关注和进一步的研究。

关键词：三环氟喹诺酮三嗪抗菌活性抗细胞增殖活性

Design, synthesis, antibacterial and anti-cell proliferation activities of [1,2,4]triazino[3,4-h][1,8]naphthyridine-8-one-7-carboxylic acid derivatives

GAO Liu-zhou¹, LI Tao¹, XIE Yu-suo¹, HUANG Wen-long², ZHAO Hui¹, HU Guo-qiang¹

1. Institute of Chemical Biology, Henan University, Kaifeng 475001, China;
2. Centre of Drug Discovery, China Pharmaceutical University, Nanjing 210009, China

Abstract: To discover novel fluoroquinolone lead compounds as possible anti-infective or/and antitumor chemotherapies, combination principle of pharmacophore-based drug design, a series of novel tricyclic fluoroquinolone title compounds, [1,2,4]triazino[3,4-h][1,8]naphthyridine-8-one-7-carboxylic acid derivatives (5a-5p), were designed and synthesized with a fused [1,2,4]-triazine ring unit. Their structures were characterized by spectral data and elemental analysis and the in vitro antibacterial and anti-cell proliferation activities were also evaluated. The results showed that the titled compounds exhibited more significant inhibitory activities against drug-resistant bacteria (Methicillin-resistant Staphylococcus aureus and multi drug-resistant Escherichia coli strains) and three tested cancer cell lines (human hepatoma SMMC-7721, murine leukemia L1210 and human murine leukemia HL60 cells). Interestingly, SAR showed that compounds with electron-donating groups attached to benzene ring had stronger antibacterial activity than antitumor activity, but electron-withdrawing compounds displayed more potential antitumor activity than antibacterial activity, especially antitumor activity of nitro compounds was comparable to comparison doxorubicin. Thus, novel triazine-fused tricyclic fluoroquinolones as potent anti-infective or/and antitumor lead compounds are valuable to pay attention and for further development.

Key words: tricyclic fluoroquinolone triazine antibacterial activity anti-cell proliferation activity

氟喹诺酮作为临床广泛使用的广谱、高效、低毒的抗感染药物备受关注^[1]。然而, 由于目前细菌耐药的日益严重, 如何克服细菌耐药性已成为抗感染药物治疗领域亟待解决的公共卫生问题^[2]。虽然解决细菌耐药的策略有多种选择, 但根本的途径是基于现有药物的结构特征进行结构优化, 不断促进新药的产生^[3]。氟喹诺酮抗菌活性的构-效关系研究表明, 除C-3位羧基和C-4羰基是抗菌活性的必需基团外, C-7位取代基 (尤其是引入C-7哌嗪基) 对抗菌谱、抗菌活性和药代动力学均有较大影响。因此, 对C7-位取代基的化学修饰或取代基的改变是获得更优秀喹诺酮类抗菌药的重要途径之一^[4]。另一方面, 拓扑异构酶不但是氟喹诺酮抗菌作用的靶点, 同时也是抗肿瘤药物的重要作用靶点之一。为此, 基于机制的合理药物设计原理, 可转化其抗菌活性到抗肿瘤活性^[5]。研究发现, C-3位羧基虽是抗菌必需的, 但并非是抗肿瘤必要的药效团, 可被其等排体如酰腙^[6]、杂环^[7]或稠杂环^[8]替代, 但这易导致抗菌活性的减弱或消失。基于此, 如何设计发现具有抗菌和抗肿瘤活性的氟喹诺酮先导化合物, 将是一项新挑战。

作为新的尝试, 设想保留抗菌活性所需的C-3羧基, 把具有抗菌^[9]和抗肿瘤活性^[10]的[1,2,4]-三嗪杂环作为C-7哌嗪基的生物等排体, 与喹诺酮母核相稠合构筑成新三环氟喹诺酮羧酸目标化合物, 希望实现药效团的活性叠加, 发现具有抗菌抗肿瘤活性的先导化合物。通过在取代基苯环上引入不同电性和亲水性的基团, 进一步考察对活性的影响, 得出初步的构-效关系, 为更具新结构化合物的设计提供新的思路。

氟氯萘啶羧酸酯 (1) 在甲醇中与80% 水合肼同时发生取代和水解反应可得到6-氟-7-肼基-萘啶-4(1H)- 酮-3-羧酸 (2) 中间体。溴代苯乙酮类的羰基可与2的互变异构体 (3) 的C-7肼基的端位氨基先发生缩合反应形成开环亚胺 (4), 然后溴与环内胺发生分子内的取代环合反应得到三环氟喹诺酮羧酸目标化合物[3-取代苯基-5-环丙基-10-氟-4H-[1,2,4]三嗪并[3,4-h] [1,8]喹啉-8(5H)-酮-7-羧酸衍生物 (5a～5n)], 其中对-硝基目标化合物5m和间硝基化合物5n经铁粉还原到相应氨基目标化合物5o和5p。化合物的合成见合成路线1。

结果与讨论 1 化学部分

氟氯萘啶羧酸酯 (1) 在甲醇中与80% 的水合肼同时发生取代和水解反应可方便得到6-氟-7-肼基-萘啶-4(1H)-酮-3-羧酸 (2) 中间体。如改成高沸点的乙醇、丙醇、丁醇等或DMSO、DMF等作溶剂可导致产品2的外观较差, 且有较多的杂质点产生。

R: H (a); 4-CH₃O (b); 2-CH₃O (c); 3,4-(CH₃O)₂ (d); 4-OH (e); 4-CH₃ (f); 2-CH₃ (g); 4-Cl (h); 3-Cl (i); 4-Br (j); 4-F (k); 2-F (l); 4-NO₂ (m); 3-NO₂ (n); 4-NH₂ (o); 3-NH₂ (p). Scheme 1 Synthetic route of the title compounds 5a-5p

目标化合物的结构经¹H NMR、MS及元素分析确证, 其收率、物理常数及波谱数据见表 1、2。

Table 1 Physical constants and spectral data of target compounds (5a-5p)

Compd.	Yield/%	mp/℃	Elemental analysis (%, Calcd.)			MS (m/z) [M+H]⁺
Compd.	Yield/%	mp/℃	C	H	N	MS (m/z) [M+H]⁺
5a	76.5	237-239	63.72 (63.49)	3.86 (4.00)	14.93 (14.81)	379
5b	84.6	238-240	61.98 (61.76)	4.38 (4.20)	15.85 (15.67)	409
5c	71.4	215-217	61.85 (61.76)	4.03 (4.20)	15.89 (15.67)	409
5d	78.3	226-228	60.49 (60.27)	4.12 (4.37)	13.04 (12.78)	439
5e	87.5	235-237	61.13 (60.91)	3.61 (3.83)	14.43 (14.21)	395
5f	76.0	217-219	64.52 (64.28)	4.20 (4.37)	14.53 (14.28)	393
5g	72.7	203-205	64.46 (64.28)	4.24 (4.37)	14.47 (14.28)	393
5h	72.7	212-214	58.44 (58.19)	3.67 (3.42)	13.74 (13.57)	413
5i	74.5	205-207	58.45 (58.19)	3.17 (3.42)	13.83 (13.57)	413
5j	86.2	226-228	52.73 (52.54)	2.87 (3.09)	12.48 (12.25)	457, 459
5k	88.4	246-248	60.84 (60.61)	3.44 (3.56)	14.37 (14.14)	397
5l	81.6	225-227	60.86 (60.61)	3.37 (3.56)	14.31 (14.14)	397
5m	90.5	256-258	56.93 (56.74)	3.51 (3.33)	16.74 (16.54)	424
5n	85.7	244-246	56.92 (56.74)	3.13 (3.33)	16.78 (16.54)	424
5o	63.2	216-218	61.29 (61.07)	3.93 (4.10)	18.05 (17.80)	394
5p	53.8	204-206	61.32 (61.07)	4.27 (4.10)	17.96 (17.80)	394

Table 1 Physical constants and spectral data of target compounds (5a-5p)

Table 2 ¹H NMR data of the target compounds (5a-5p)

Compd.	¹H NMR (400 MHz, DMSO-d₆)
5a	1.15-1.24 (m, 4H, 2'-and 3'-H), 5.16 (s, 2H, 4-H), 7.16-7.63 (m, 5H, Ph-H), 7.86 (d, J = 13.2 Hz, 8-H), 8.88 (s, 1H, 6-H)
5b	1.17-1.26 (m, 4H, 2'-and 3'-H), 3.87 (s, 3H, OCH₃), 5.18 (s, 2H, 4-H), 7.45 (d, J = 7.4 Hz, 2H, Ph-H), 7.68 (d, J = 7.4 Hz, 2H, Ph-H), 7.87 (d, J = 13.2 Hz, 9-H), 8.89 (s, 1H, 6-H)
5c	1.21-1.28 (m, 4H, 2'-and 3'-H), 3.86 (s, 3H, OCH₃), 5.16 (s, 2H, 4-H), 7.42-7.67 (m, 4H, Ph-H), 7.88 (d, J = 13.2 Hz, 9-H), 8.87 (s, 1H, 6-H)
5d	1.18-1.32 (m, 4H, 2'-and 3'-H), 3.86, 3.88 (s, 6H, 2×OCH₃), 5.16 (s, 2H, 4-H), 7.45-7.64 (m, 3H, Ph-H), 7.87 (d, J = 13.2 Hz, 9-H), 8.89 (s, 1H, 6-H)
5e	1.22-1.28 (m, 4H, 2'-and 3'-H), 5.18 (s, 2H, 4-H), 7.57 (d, J = 7.2 Hz, 2H, Ph-H), 7.72 (d, J = 7.2 Hz, 2H, Ph-H), 7.86 (d, J = 13.2 Hz, 9-H), 8.92 (s, 1H, 6-H), 9.68 (br s, 1H, OH)
5f	1.16-1.25 (m, 4H, 2'-and 3'-H), 2.34 (s, 3H, CH₃), 5.14 (s, 2H, 4-H), 7.44 (d, J = 7.2 Hz, 2H, Ph-H), 7.62 (d, J = 7.2 Hz, 2H, Ph-H), 7.87 (d, J = 13.2 Hz, 9-H), 8.84 (s, 1H, 6-H)
5g	1.15-1.26 (m, 4H, 2'-and 3'-H), 2.28 (s, 3H, CH₃), 5.15 (s, 2H, 4-H), 7.38-7.63 (m, 4H, Ph-H), 7.86 (d, J = 13.2 Hz, 9-H), 8.86 (s, 1H, 6-H)
5h	1.21-1.28 (m, 4H, 2'-and 3'-H), 5.18 (s, 2H, 4-H), 7.56 (d, J = 7.5 Hz, 2H, Ph-H), 7.73 (d, J = 7.5 Hz, 2H, Ph-H), 7.88 (d, J = 13.2 Hz, 9-H), 8.92 (s, 1H, 6-H)
5i	1.23-1.31 (m, 4H, 2'-and 3'-H), 5.18 (s, 2H, 4-H), 7.58-7.74 (m, 4H, Ph-H), 7.87 (d, J = 13.2 Hz, 9-H), 8.90 (s, 1H, 6-H)
5j	1.18-1.26 (m, 4H, 2'-and 3'-H), 5.15 (s, 2H, 4-H), 7.43 (d, J = 7.5 Hz, 2H, Ph-H), 7.68 (d, J = 7.5 Hz, 2H, Ph-H), 7.86 (d, J = 13.2 Hz, 9-H), 8.89 (s, 1H, 6-H)
5k	1.24-1.34 (m, 4H, 2'-and 3'-H), 5.21 (s, 2H, 4-H), 7.63 (d, J = 7.5 Hz, 2H, Ph-H), 7.78 (d, J = 7.5 Hz, 2H, Ph-H), 7.91 (d, J = 13.2 Hz, 9-H), 8.92 (s, 1H, 6-H)
5l	1.23-1.33 (m, 4H, 2'-and 3'-H), 5.22 (s, 2H, 4-H), 7.62-7.78 (m, 4H, Ph-H), 7.87 (d, J = 13.2 Hz, 9-H), 8.90 (s, 1H, 6-H)
5m	1.24-1.37 (m, 4H, 2'-and 3'-H), 5.25 (s, 2H, 4-H), 7.68 (d, J = 7.5 Hz, 2H, Ph-H), 7.82 (d, J = 7.5 Hz, 2H, Ph-H), 7.91 (d, J = 13.2 Hz, 9-H), 8.95 (s, 1H, 6-H)
3n	1.22-1.35 (m, 4H, 2'-and 3'-H), 5.24 (s, 2H, 4-H), 7.66-7.80 (m, 4H, Ph-H), 7.89 (d, J = 13.2 Hz, 9-H), 8.93 (s, 1H, 6-H)
5o	1.18-1.27 (m, 4H, 2'-and 3'-H), 5.17 (s, 2H, 4-H), 7.46 (br s, 2H, NH₂), 7.56 (d, J = 7.4 Hz, 2H, Ph-H), 7.68 (d, J = 7.4 Hz, 2H, Ph-H), 7.87 (d, J = 13.2 Hz, 9-H), 8.86 (s, 1H, 6-H)
5p	1.17-1.30 (m, 4H, 2'-and 3'-H), 5.18 (s, 2H, 4-H), 7.47-7.66 (m, 6H, NH₂ and Ph-H), 7.88 (d, J = 13.2 Hz, 9-H), 8.87 (s, 1H, 6-H)

Table 2 ¹H NMR data of the target compounds (5a-5p)

2 抗菌活性和抗肿瘤活性评价

用标准试管二倍稀释技术测定了目标化合物体外对革兰氏阳性金葡菌 (Staphylococcus aureus ATCC 29213)、革兰氏阴性埃希氏大肠杆菌(Escherichia coli ATCC 29213) 及耐甲氧西林金葡菌 (methicillin- resistant Staphylococcus aureus ATCC 25923) 和多药耐药埃希氏大肠杆菌 (multi-drug resistant Escherichia coli ATCC 35218) 的最低抑菌浓度 (MIC), 并与对照品环丙沙星相比较, 依此评价其抗菌活性。抗菌实验结果 (表 3) 表明, 虽然目标化合物对标准阳性金葡菌及阴性大肠杆菌的抗菌活性弱于对照环丙沙星的活性, 但其对耐药菌株, 尤其是对多药耐药大肠杆菌抑制活性明显强于环丙沙星。苯环带有供电子基如羟基 (5e)、氨基 (5o、5p) 或F原子 (5k、5l) 等化合物对耐药菌株的活性最敏感, 这为以后的结构优化提供了重要信息。

Table 3 Inhibitory activities against microbes (MIC, μg·mL^-1) and tested tumor cell lines (IC₅₀, μmol·L^-1) for the title compounds (5a-5p) (n = 3). ^aStaphylococcus aureus ATCC 29213, ^bEscherichia coli ATCC 29213, ^cmethicillin-resistant Staphylococcus aureus ATCC 25923, ^dmulti-drug resistant Escherichia coli ATCC 35218

Compd.	Inhibitory activities against microbes (MIC) /μg·mL^-1				Inhibitory activities against tumor cell lines (IC₅₀) /μmol·L^-1
Compd.	S. aureus^a	E. coli^b	MR-S. aureus^c	MDR-E. coli^d	SMMC-7721	L1210	HL60
5a	4.0	4.0	8.0	4.0	15.3 ± 1.24	28.7 ± 2.76	32.7 ± 2.68
5b	8.0	2.0	1.0	0.5	17.8 ± 1.34	25.6 ± 1.40	28.6 ± 1.27
5c	1.0	1.0	0.5	0.25	18.4 ± 1.56	23.8 ± 1.58	21.3 ± 2.10
5d	8.0	2.0	2.0	1.0	32.7 ± 2.72	41.7 ± 3.52	43.4 ± 3.16
5e	1.0	0.5	0.5	0.5	6.7 ± 0.43	10.6 ± 1.40	15.7 ± 1.24
5f	32.0	32.0	8.0	2.0	32.0 ± 2.60	38.4 ± 2.44	46.8 ± 3.24
5g	1.0	32.0	8.0	1.0	37.0 ± 3.15	41.6 ± 3.82	38.5 ± 3.36
5h	8.0	4.0	4.0	2.0	38.6 ± 3.20	36.5 ± 1.73	45.7 ± 3.72
5i	4.0	4.0	8.0	2.0	35.7 ± 2.63	40.7 ± 2.20	48.6 ± 2.75
5j	16.0	8.0	8.0	4.0	1.8 ± 0.14	7.8 ± 0.43	12.5 ± 1.07
5k	0.5	0.5	0.25	0.25	1.3 ± 0.12	8.4 ± 1.13	15.7 ± 1.32
5l	0.5	1.0	0.25	0.25	2.4 ± 0.18	6.7 ± 0.54	10.4 ± 0.53
5m	32.0	16.0	8.0	4.0	1.0 ± 0.12	3.5 ± 0.46	8.6 ± 0.42
5n	32.0	32.0	8.0	4.0	1.6 ± 0.15	3.2 ± 0.28	5.7 ± 0.32
5o	1.0	0.5	0.5	0.25	35.7 ± 3.12	36.7 ± 2.80	41.6 ± 3.16
5p	1.0	0.5	0.5	0.25	28.6 ± 2.40	33.4 ± 2.38	38.9 ± 2.46
Ciprofloxacin	0.25	0.25	4.0	8.0	≥ 100	≥ 100	≥ 100
Doxorubicin	-	-	-	-	2.3 ± 0.26	4.2 ± 0.35	3.5 ± 0.27

采用MTT方法测定了目标化合物对人肝癌细胞 (SMMC-7721)、鼠白血病细胞 (L1210) 和人白血病细胞 (HL60) 的半数抑制浓度 (IC₅₀)。结果 (表 3) 表明, 16个目标化合物的IC₅₀值均低于50.0 μmol·L^-1, 显示出潜在的抗肿瘤活性。尤其是羟基、F原子取代的苯环能增强抗肿瘤活性, 这与抗菌活性的构-效关系一致。意外的是, 硝基化合物转化为氨基化合物, 其抗肿瘤活性显著下降, 如硝基化合物5m和5n对肝癌细胞SMMC-7721的抑制活性与对照蒽醌类阿霉素的活性相当, 是氨基化合物5o和5p活性 (IC₅₀值) 的5～40倍, 具有进一步研究的价值。

为此, 三嗪稠合的三环氟喹诺酮为抗菌抗肿瘤先导化合物的设计提供了新思路。

实验部分

熔点测定采用WK-1B数字熔点仪 (上海精密科学仪器厂), 毛细管法, 温度未校正; AM-400型核磁共振仪 (德国Bruker公司), DMSO-d₆为溶剂; Esquire LC型质谱仪 (德国Bruker公司); 2400-Ⅱ元素分析仪 (美国PE公司)。实验菌株由河南大学淮河临床医院检验科提供, 并负责筛选; 肿瘤细胞购自中国科学院细胞库。氟氯萘啶羧酸乙酯1为市售品, 所用试剂均为分析纯。

1 化学合成 1.1 中间体1-环丙基-6-氟-7-肼基-[1,8]萘啶-4(1H)-酮-3-羧酸 (2) 的合成

氟氯萘啶羧酸乙酯1 (20.0 g, 70.8 mmol) 与80% 的水合肼 (7.0 g, 140.0 mmol) 在无水甲醇 (300 mL) 中搅拌回流反应12 h。冷却到室温过滤产生的固体, 用水洗涤。粗品直接用稀盐酸(3.6%, 200 mL) 溶解, 加适量的活性炭回流脱色1 h。热过滤, 滤液用氨水中和至pH 7.0, 滤集生成的固体, 水洗, 干燥, 得淡黄色固体2, 收率75.0%, mp 232～234 ℃; MS (m/z): 279 [M+H]⁺; Calcd. for C₁₂H₁₁FN₄O₃: 278.24。

1.2 3-苯基-5-环丙基-10-氟-4H-[1,2,4]三嗪并[3,4-h] [1,8]萘啶-8(5H)-酮-7-羧酸 (5a-5n) 合成通法 (以5a为例)

肼基氟萘啶羧酸2 (0.5 g, 1.8 mmol) 与无水乙酸钠 (0.22 g, 2.7 mmol) 溶解于冰乙酸 (5 mL)中, 加入溴代苯乙酮 (0.36 g, 1.8 mmol)。混合物搅拌回流反应6 h, 减压蒸除溶剂, 残留物用无水乙醇-DMF混合溶剂溶解, 加适量的活性炭脱色30 min, 热过滤除去生成的溴化钠, 放置过夜。滤集析出的固体, 用无水乙醇洗涤, 干燥, 得目标物5a。用溴代苯乙酮的衍生物替代上述的溴代苯乙酮, 按同法制得目标物5b～5n。

1.3 3-(对氨基苯基/间氨基苯基)-5-环丙基-10-氟- 4H-[1,2,4]三嗪并[4,3-h][1,8]萘啶-8(10H)-酮-7-羧酸 (5o/5p) 的合成

将铁粉 (1.0 g, 18.0 mmol)、浓盐酸 (1 mL) 与95% 乙醇 (50 mL) 的混合物搅拌回流10 min, 加入上述制备的硝基化合物5m/5n (1.0 g, 3.0 mmol), 回流反应至原料消失。热过滤, 蒸除溶剂乙醇, 加水分散固体, 用氨水调中性。滤集产生的固体, 水洗, 干燥。粗品用乙醇-DMF重结晶, 得相应的氨基化合物 (5o/5p)。

2 生物活性评价 2.1 抗菌活性评价^[11]

将革兰氏阳性金葡菌 (Staphylococcus aureus ATCC 29213)、革兰氏阴性埃希氏大肠杆菌(Escherichia coli ATCC 29213) 及耐甲氧西林金葡菌 (methicillin-resistant Staphylococcus aureus ATCC 25923) 和多药耐药埃希氏大肠杆菌(multi-drug resistant Escherichia coli ATCC 35218) 分别接种于pH 8.0、经过灭菌的MH肉汤培养基2.0 mL, 于37 ℃培养18 h, 并用MH肉汤稀释成1/200, 作为试验菌液备用。采用试管二倍稀释方法, 将供试目标化合物5a～5p和对照药环丙沙星用DMSO配成 128 μg·mL^-1浓度的储备液, 以二倍递减浓度稀释, 分别加入上述备用菌液0.1 mL, 于37 ℃培养18 h, 肉眼观察其生长情况, 无细菌生长的浓度即为最低抑菌浓度 (MIC), 并与对照药环丙沙星相比较, 评价其抗菌活性。

2.2 抗肿瘤活性评价^[8]

16个三环氟喹诺酮目标化合物及对照药环丙沙星和结构类似抗癌药物阿霉素用DMSO配成1.0×10^-2 μmol∙L^-1浓度的储备液, 用RPMI-1640稀释到所需浓度。取对数生长期的人肝癌SMMC-7721细胞、鼠白血病L1210细胞和人白血病HL60细胞分别以每孔7 000个细胞接种于96孔板, 培养隔夜后, 加入不同浓度的上述供试化合物溶液, 继续培养48 h后弃去培养基。每孔加入1 g·L^-1 MTT溶液100 μL, 继续培养4 h后弃上清液。每孔加入150 μL二甲基亚砜, 轻轻振荡30 min, 用酶标仪在570 nm波长处测其吸光度值。计算各组对癌细胞的抑制率: 抑制率% = [(1-实验组吸光度值) / 对照组吸光度值] ×100%。然后以各药物浓度对数值对各浓度下的抑制率作线性回归, 得浓度-效应方程, 以此计算出各供试化合物对实验癌细胞的半数抑制浓度 (IC₅₀)。

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药学学报 2015, Vol. 50 Issue (3): 332-336	PDF